Structure of GDAP1 bound to a product of lipid peroxidation

摘要 GDAP 1突变与周围神经病Charcot-Marie-Tooth病相关沙尔科马 裂齿症是最常见的遗传性神经系统疾病之一，据估计， 只有美国。GDAP 1敲低、过表达以及多种CMT模型显示了变化， 与细胞对氧化应激反应的失调一致。这些变化包括NRF 2驱动的 转录活性、谷胱甘肽耗竭、氧化还原失衡和线粒体去极化的证据。在 与此同时，线粒体网络对氧化应激反应的关键方面非常类似于 CMT表型的方面：片段化、融合缺陷和细胞内位置的变化。最后，GDAP 1 是谷胱甘肽S-转移酶（GST）超家族的成员，其保护细胞免受过氧化脂质的侵害 和外源性物质，即在氧化应激条件下积累的有毒分子。其背后的机制- GDAP 1在氧化应激反应中的作用尚不清楚。我们最近发现GDAP 1可以结合 4-羟基壬烯醛（4 HNE）是一种有毒脂质，主要由体内的过氧化脂质分解形成。 线粒体该提案将解决两个主要问题：GDAP 1是否可以利用4 HNE作为底物， 方式类似于典型的GST酶，或者它在氧化应激反应中具有非酶促作用， sponse？其次，是否存在与4 HNE结合相关的构象变化或4 HNE结合的结果？我们 将通过1）生物化学测量与4 HNE介质相关的酶参数来解决这些问题， 2）生物化学定义推定活性位点口袋内重要残基的影响 对4 HNE结合亲和力和GST酶活性的影响; 3）确定GDAP 1 - 4 HNE复合物的结构 使用X射线晶体学。这些数据将定义GDAP 1识别4 HNE的分子机制 以促进结合并揭示与4 HNE结合相关的蛋白质中的构象变化。如果 GDAP 1是一种酶，它将揭示它如何促进谷胱甘肽催化4 HNE。如果GDAP 1正在播放 非酶促作用，4 HNE引起的构象变化将在GDAP 1功能中发挥关键作用， 在这个结构中显露出来。总的来说，这些研究将是至关重要的，在塑造未来的生化和细胞为基础的 以GDAP 1功能为中心的研究。此外，该结构将提供计算所需的基础， 静态预测小分子结合伴侣，调节GDAP 1功能的关键工具， 这是对CMT疾病模型的更深入研究，也是迈向治疗干预的第一步。

Summary. Mutations in GDAP1 are associated with the peripheral neuropathy Charcot-Marie-Tooth disease. Charcot-Ma- rie-Tooth is one of most common inherited neurological disorders, estimated to affect 126,000 people in the United States alone. GDAP1 knockdown, overexpression, and as well as multiple models of CMT show changes consistent with dysregulation of the cellular response to oxidative stress. These include changes in NRF2-driven transcriptional activity, evidence of glutathione depletion, redox disbalance, and mitochondrial depolarization. At the same time key aspects of the mitochondrial network’s response to oxidative stress are very similar to key aspects of CMT phenotypes: fragmentation, fusion deficits and change in position inside the cell. Finally, GDAP1 is a member of the Glutathione S-transferase (GST) superfamily which protect the cell against peroxidated lipids and xenobiotics, toxic molecules that accumulate under conditions of oxidative stress. The mechanism underly- ing GDAP1’s role in oxidative stress response is unknown. We have recently discovered that GDAP1 can bind 4-hydroxynoneal (4HNE), a toxic lipid that is formed from the breakdown of peroxidated lipids primarily in the mitochondria. This proposal will address two main questions: can GDAP1 utilize 4HNE as a substrate, in a manner similar to canonical GST enzymes, or does it have a non-enzymatic role in the oxidative stress re- sponse? Secondly, are there conformational changes associated with or a consequence of 4HNE binding? We will address these questions by 1) biochemically measuring enzymatic parameters associated with 4HNE medi- ated GST activity; 2) biochemically defining the impact of important residues within the putative active site pocket on 4HNE binding affinity and GST enzymatic activity; 3) determining the structure of the GDAP1-4HNE complex using x-ray crystallography. These data will define the molecular mechanism by which GDAP1 recognizes 4HNE to facilitate binding and reveal and conformational changes in protein that are associated with 4HNE binding. If GDAP1 is an enzyme it will reveal how it facilitates catalysis of 4HNE with glutathione. If GDAP1 is playing a non-enzymatic role, conformational changes resulting from 4HNE will play a key role in GDAP1 function and will be revealed in this structure. Overall, these studies will be critical in shaping future biochemical and cell-based investigations centered on GDAP1 function. Further, the structure will provide the foundation needed to compu- tationally predict small molecule binding partners, critical tools for modulating GDAP1 function that will allow deeper interrogation of CMT disease models and a first step towards therapeutic intervention.